提高bFGF基因表达水平的策略及其表达产物的纯化工艺  

Strategy on Enhancement of bFGF Gene Expression and Purification of Its Products

作　　者：王莉馨[1] 刘雅娟[2] 林剑[3] 范洪学[2] 金宁一[1] 

机构地区：[1]解放军军需大学病毒研究所,吉林长春130021 [2]吉林大学预防医学院 [3]广州暨南大学生物工程研究所

出　　处：《中国肿瘤生物治疗杂志》2001年第2期142-142,共1页Chinese Journal of Cancer Biotherapy

基　　金：国家九五科技攻关项目 ( 96 C0 2 0 1 0 3)资助

摘　　要：目的 :通过在同一个表达载体pLX1上调整SD序列和ATG之间距离 ,以提高bFGF目的蛋白表达量。方法 :以Klenow和MungBeanNuclease通过增加 2个及减少 2个碱基调整SD序列和ATG之间距离 ,SDS PAGE和Westernblot检测目的蛋白表达量 ,以高压液相疏水层析、凝胶过滤及肝素亲和层析纯化目的蛋白、MTT法进行活性测定。结果 :获得了重组质粒pLX2 ,pLX3 ,诱导后与表达质粒pLX1( 7.78% )相比 ,表达量分别为 8.0 3 % ,9.9% ,经纯化后获得的bFGF纯度达 98% ,ED50 为 2 .2 9ng/ml。结论 :调整SD序列和ATG之间距离可以增加目的基因的剂量 。Objective: To adjust the distance between SD sequence and ATG in the same expressive plasmid pLX1 to enhance expression of heterologous bFGF gene in E. coli. Methods: Adjusting the different distance between SD sequence and ATG by Klenow and Mung Bean Nuclease. SDS PAGE and Western blot showed the expressed protein bFGF in E.coli. bFGF proteins were purified by HPHIC, HPGFC and HAC. Biological activity was examined by MTT. Results: Recombinant plasmids pLX2, pLX3 were obtained and the expressive levels were 8.03%, 9.9% respectively. Also the purified bFGF was obtained by HPHIC, HPGFC, HAC and its ED 50 was 2.29 ng/ml. Conclusion: Increasing the bFGF gene dosage by adjusting the distance between SD sequence and ATG could increase the expression level of a desired protein.

关 键 词：BFGF ATG SD序列 基因表达 纯化 碱性成纤维细胞因子 

分 类 号：R392.2[医药卫生—免疫学]